This project is centered on the mechanisms of exocytosis, the ubiquitous eukaryotic process by which vesicles fuse to the plasma membrane and release their contents. We report two subprojects this year:[unreadable] [unreadable] 1. The mechanism of synaptic release. One of the abiding mysteries in biology is the great speed of synaptic transmission, where synaptic vesicles laden with neurotransmitter fuse to the presynaptic membrane and release their content to the synaptic cleft. The Ca2+ - triggered release of neurotransmitter begins some tens of microseconds after Ca2+ floods the presynaptic intracellular release site. Thus the mechanism of membrane fusion must account for how Ca2+ triggers the extremely fast formation of a fusion pore linking vesicular and plasma membranes that were hitherto stable and non-leaky. The current paradigm for exocytotic fusion sets the trans-SNARE complex, composed of proteins localized in vesicular and plasma membranes, as the minimal fusion machine. The calcium-dependence of fusion is believed to be regulated by proteins such as synaptotagmin 1, which acts as both ?calcium sensor? mediating Ca2+-triggering and regulator of fusion pore dynamics during neurotransmitter release. But synaptotagmin 1 may have a more central role in mediating fast synaptic fusion.[unreadable] [unreadable] We propose that ring assemblies of SNAREs and synaptotagmin complexes form to appropriately concentrate and orient C2b domains of synaptotagmin. The ordered domains then create an electrostatictunnel for membrane fusion5 that is extended by the polybasic linker regions of syntaxin and synaptobrevin. What is the role of calcium? First, Ca2+ turns on an ?electrostatic switch? initially proposed for synaptotagmin-syntaxin interaction, but better suited to instantaneously stress the phospholipid bilayers of the presynaptic membrane and the synaptic vesicle for the ultra-rapid exocytosis seen in the nervous system. Second, even without synaptotagmin Ca2+ speeds up fusion of SNARE-reconstituted membranes considerably. Perhaps Ca2+ plays a direct role, electrostatically complexing PS headgroups to promote fusion between negatively charged phospholipid bilayers. [unreadable] [unreadable] Ultimately, synaptotagmin, SNAREs, and the other proteins that comprise the exocytotic fusion machine must cajole lipids to move through a pathway that culminates in fusion pore opening., Our view is that exocytotic fusion follows the pathway of phospholipid membrane fusion. The role of proteins along this pathway is to lower the several energy barriers to membrane fusion, just as enzymes lower the energy barriers to their respective reactions. Since the reaction coordinate for membrane fusion is the radius of the stalk and pore proteins controlling radial forces should regulate forward and backward passage through the pathway towards complete fusion. The SNARE proteins and synaptotagmin are the guides that walk and pull the membrane through a bumpy stalk-pore path, with electrostatic interactions playing a larger role than hitherto realized. [unreadable] [unreadable] 2. The creation of macroscopic raft domains in lipid membranes. We describe quantitatively the creation and evolution of phase-separated domains in a multicomponent lipid bilayer membrane. The early stages, termed the nucleation stage, and the independent growth stage, are extremely rapid (characteristic times are submillisecond and millisecond, respectively) and the system consists of nanodomains of average radius about 5 -50 nm. Next, mobility of domains becomes consequential; domain merger and fission become the dominant mechanisms of matter exchange, and line tension is the main determinant of the domain size distribution at any point in time. For sufficiently small line tension, the decrease in the entropy term that results from domain merger is larger than the decrease in boundary energy, and only nanodomains are present. For large line tension, the decrease in boundary energy dominates the unfavorable entropy of merger, and merger leads to rapid enlargement of nanodomains to radii of micrometer scale. At intermediate line tensions and within finite times, nanodomains can remain dispersed and coexist with a new global phase. The theoretical critical value of line tension needed to rapidly form large rafts is in accord with the experimental estimate from the curvatures of budding domains in giant unilamellar vesicles.